Alignment of CNC family representatives from metazoans has shown that there was one ancestral CNC family gene that has been duplicated more than once in the lineage leading to the chordates (Grimberg et al., 2011). There is only one CNC family member present in arthropod taxa (Mohler et al., 1991) and one present in the nematode C. elegans, Skn-1 (Bowerman et al., 1992; Walker et al., 2000). Four homologous CNC genes are located 3’ to the four Hox clusters of vertebrates: NF-E2 (Andrews et al., 1993), Nrf1 also known as LCR-F1 in mice (Chan et al., 1993; Caterina et al., 1994; Chan et al., 1996; Farmer et al., 1997), Nrf2 or ECH (Moi et al., 1994; Itoh et al., 1995), and Nrf3 (Kobayashi et al., 1999; Etchevers, 2005). Functions of CNC family members are diverse, but often include conserved functions such as the specification of midgut and pharyngeal tissue, roles in hematopoiesis, and xenobiotic and oxidative stress responses in addition to the developmental functions present in arthropods (Blackwell et al., 1994; Itoh et al., 1997; Alam et al., 1999; Wild et al., 1999;

Walker et al., 2000; Grimberg et al., 2011). Despite the proximity of CNC family members to Hox gene clusters, no interaction between CNC homologues and Hox genes has been observed outside of arthropods (Etchevers, 2005).

In order to investigate the primitive functions of the mandibular patterning genes cnc and Dfd, it is necessary to study their expression and function in groups outside of the mandibulates. The function, and indeed expression of cnc is unknown in non-mandibulate arthropods. Prior to this current investigation, it was not known whether there was a cnc orthologue present in any non-mandibulate arthropod taxa.

The only extant non-mandibulate arthropod group is the Chelicerata, which includes the arachnids (spiders, scorpions, ticks, mites and harvestmen) and xiphosurans (horseshoe crabs). Less closely related taxa (present in Panarthropoda) include the Onychophora (velved worms) and the Tardigrada (Brusca and Brusca, 2003; Budd and Telford, 2009).

The homologous segment to the mandibular segment in chelicerates is the first walking leg segment. The strongest evidence for this comes from examination of the anterior boundary of Hox gene expression. Comparison of the anterior limit of Hox gene expression in mandibulates reveals that the expression domains are conserved.

For example, the anterior limit of labial and proboscipedia is present in the first postantennal segment which is the second antennal segment of crustaceans and the intercalary segment of myriapods and hexapods. The anterior boundary of Dfd is present in the second post-antennal segment which is the mandibular segment of all mandibulates (Hughes and Kaufman, 2002a; Scholtz and Edgecombe, 2006).

Comparison of anterior Hox gene expression domains of lab, pb, Hox3, Dfd and scr in the spiders Cupiennius salei, Achaearanea, Steatoda triangulosa and the orbatid mite Archegozetes longisetosus enabled segmental homologies to be inferred between the chelicerates and the mandibulates. The chelicerae bearing segment is homologous to the antennal segment, as there are no Hox genes expressed in these segments. The first post-cheliceral segment, the pedipalp segment, is homologous to the second antenna/intercalary segment (marked by the anterior boundary of lab and pb). The second post-cheliceral/antennal segment, the first leg segment, is homologous to the mandibular segment and is marked by the anterior boundary of Dfd expression (Damen et al., 1998; Telford and Thomas, 1998b; Abzhanov and Kaufman, 1999a;

Schwager et al., 2007). The segmental expression of the full complement of Hox genes of the spider Cupiennius salei is shown in fig.6.1.

Fig.6.1. Segmental expression patterns of the ten Hox genes of Cupiennius salei. Anterior is to the left.

Segments are in anterior to posterior order: ocular (Oc), chelicerae (Ch), pedipalps (Ped) and four Leg segments (L1-L4) opisthosomal segments (O). The Prosoma/opisthosoma boundary is indicated with a dotted line. wg and en expression marks the posterior of each segment. There are duplications of Hox genes Dfd, Scr and Ubx. The homologous segment to the mandibular segment is the L1 segment. The L1 segment has lab, pb, Hox3 and Dfd expression. The anterior expression boundary of Dfd, (which may relate to its function) is expressed in the L1. Note the posterior boundary of the expression domains of anterior Hox genes lab, pb, Hox3, Dfd, Scr and ftz extend to the most posterior prosomal segment the L4. The expression of the posterior Hox genes Antp, Ubx, abdA, AbdB extends to the posterior opisthosomal segment (O12). Figure from (Schwager et al., 2007).

Outside of Chelicerata and Mandibulata, expression of lab, pb and Dfd in a pycnogonid Endeis spinosa homologises the mandibular segment to the third pair of larval appendages (Jager et al., 2006). Study of the expression of Hox genes in an onychophoran Euperipatoides homologises the mandibular segment to the first walking leg of this species (Eriksson et al., 2010).

The legs of chelicerates present on the L1 to L4 segments are nearly identical to one another. The chelicerate L1 to L4 segments are homologous to the mandible, maxilla, labial and first thoracic segments of insects, respectively, which are clearly differentiated from each other. In Tribolium, differentiation of these four segments is accomplished by the Hox genes Tc Dfd, Cx, mxp and prothorax-less (ptl) (the Tribolium orthologue of antennapedia) (Brown et al., 2000; Shippy et al., 2000b; Curtis et al., 2001; DeCamillis et al., 2001; Brown et al., 2002a). There is no Hox gene that differentiates the mandibular segment from the maxillary segment. Instead, this is accomplished by the bZIP gene cnc (as shown in chapter four). In chelicerates, there has been no functional study of the function of anterior Hox genes. To date only one Hox gene, antp, has been studied functionally. antp represses appendage formation in the first opisthosomal segment which is the anterior-most segment where antp is expressed (Prpic, personal communication). There have been duplications of some Hox genes in some chelicerates, such as Dfd, Ubx, and Scr (Abzhanov et al., 1999; Schwager et al., 2007).

There are numerous Hox gene candidates for differentiating the second and third post-cheliceral appendages of chelicerates (the first and second leg appendages) based on the anterior limit of their expression domains. The anterior boundary of the two homologues of Dfd are expressed in the L1 segment and the anterior boundary of two hox genes Scr and ftz is present in the third post-antennal segment.

Considering that the first leg segment of chelicerates is not differentiated in any obvious morphological manner from the second leg segment, and there are numerous Hox genes that could potentially perform this role, it seems that there would be no requirement for cnc to differentiate this segment from the second leg segment. It would therefore be of interest to study cnc in a chelicerate to find out exactly what role, if any, there is of the posterior coller domain present in the mandibular segment of mandibulate arthropods which is homologous to the first leg segment of chelicerates. If there was no posterior domain of cnc present in the chelicerates, it would provide evidence that this domain has been acquired in the ancestor to mandibulates.

In Tribolium, Tc cnc differentiates the mandible from a maxilla and represses the Hox gene Tc Dfd. In all studied chelicerates, Dfd is expressed in a broad domain from the first leg segment, the homologous segment to the mandibular segment, to the fourth leg segment. It would also be interesting to study the interaction between spider homologues of cnc and Dfd, to determine whether there is any genetic interaction between cnc and Dfd (or any other Hox gene), such as present in Tribolium or whether this interaction evolved in the lineage leading to the mandibulates.

All non-mandibulate groups, the Chelicerata, Onychophora and Tardigrada are potentially informative of the ancestral function of cnc in the ancestor to mandibulate arthropods. However, onychophorans and tardigrades are harder to study (if at all in the case of the tardigrades), with no means of attempting functional genetic analysis and are less closely related to mandibulate arthropods. Chelicerates, on the other hand, are the sister group of the mandibulate arthropods and have the potential for functional genetic analyses. Developmental data comparing the development of the homologous segment of the second post-cheliceral/antennal appendage could also provide evidence to favour either of the competing phylogenetic hypotheses of the Mandibulata and the Myriochelata.

I therefore chose a suitable chelicerate to study the function of a nonmandibulate orthologue of cnc, the house spider Achaearanea tepidariorum15. The embryonic development of Achaearanea has been studied in the laboratory for several years, and so techniques such as in situ hybridization and RNAi are now routinely used to investigate its development (McGregor et al., 2008).16 The expression of the genes At Dll, At en and At Dfd-1 were studied both as controls for in situ hybridization experiments and to study the developing spider embryo.

Achaearanea tepidariorum has recently been reclassified as Parasteatoda tepidariorum. As there is an inherent inertia in the literature to reclassify familiar terminology and the majority of studies still refer to Achaearanea and not Parasteatoda, this convention will also be followed here.

Although, RNAi is somewhat problematic and feasibly limited to studying the function of early expressed developmental genes.

6.2 Results Cloning the At cnc orthologue In order to clone the homologue of cnc in Achaearanea, degenerate primers were designed against conserved regions of the cnc gene locus. Nested PCR using degenerate PCRs was performed to increase both specificity and yield of the amplified product. A PCR reaction was performed using the degenerate primer sequences based on the amino acid sequence SRDEKRA and KRKLDQI for the forward and reverse primers respectively. Amplification of the product of the first PCR of outer primer pairs was performed with nested degenerate primers based on the amino acid sequence PIDEFNE and KVAAQN for the forward and reverse primers respectively. The nested PCR reaction produced a 110bp product of which 71bp was non-primer based amplified sequence that was aligned to other arthropod homologues of cnc (see fig.6.2).

Fig.6.2. Degenerate primers used to amplify homologue of cnc in Achaearanea. Nested PCR was performed with two pairs of degenerate primers: forward outer primer SRDEKRA and reverse outer primer RKRKLDQI were used to amplify a DNA template for the nested PCR reaction. The forward nested primer PIDEFNE and the reverse nested primer KVAAQN were used to amplify a 110 bp product of which 71bp did not include the primer sequence (shown as two dark blue lines). Outer primers are indicated with blue arrows, nested primers are indicated with red arrows. The numbers indicate the nucleotide position of the 5’end of the primer from the position of the first forward primer.

The alignment of this 71bp sequence confirmed the identity of this gene fragment as a cnc related gene. The protein alignment of this region is shown in fig.6.3.

This sequence of At cnc DNA was extended by rapid amplification of cDNA ends (RACE). Two sequences were obtained from RACE, a 596bp sequence from the combined products of 3’ and 5’ RACE cloning and an additional 714bp sequence from 3’RACE cloning of the 596bp product. The combined total unique sequence of At cnc obtained was 1207bp. Both sequences were transcribed to synthesize two labelled anti-sense RNA probes to detect At cnc by in in situ hybridization. The protein alignment of the bZIP domain of At cnc to other CNC family members and bZIP family of transcription factors is shown in fig.6.4. At cnc possesses the basic DNA binding Fig.6.3. Alignment of translated 71bp sequence obtained by degenerate PCR with cnc orthologues from other arthropod taxa confirms identity as a CNC family member in Achaearanea. Figure shows a 23 amino acid alignment with half of the basic region of the bZIP domain present at the C-terminal end of the alignment. The At cnc sequence is more similar to other chelicerates Ixodes and Rhipicephalus than other arthropods.

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